This review paper aims at outlining the principle of two dimensional (2D) IR spectroscopy and its applications to the subtle structural changes of proteins in aqueous solutions. Our studies have dual purposes. One is to show that 2D ATR/IR spectroscopy can be utilized to explore the effects of adsorption of proteins on an ATR cell. Another purpose is to provide new insight into temperature and acid induced protein structure and denaturation by using 2D IR spectroscopy.